Differential growth of human enteric adenovirus 41 (TAK) in continuous cell lines.
Differing reports exist about the replication of human enteric adenoviruses (EnAds) in various cell lines. There was a suggestion that EnAds are defective, do not grow on primary human diploid cells, and behave like Ad host-range mutants, i.e., they require early gene products from other Ad types for efficient growth. Thus, initially the Graham-293 cell line, which contains the E1 region (E1A, E1B) of Ad5, was thought to be an ideal host for EnAds, because it provided the needed functions. Our findings, however, question this contention and show that Ad41 strain TAK, cultured on 293 cells, rapidly loses its infectivity (greater than 90% on the first and 100% by the second passage). In contrast to the results with 293 cells, we found that Ad41 strain TAK can be serially grown to high titers on several continuous cell lines: namely, HeLa, HI407, or HEp-2 cells. In order to investigate the basis for the rapid loss of the Ad41 infectivity upon passaging in 293 cells, Ad41 virions were purified from 293, as well as from HEp-2 cells, and their composition was analyzed. When structural proteins of the complete virions (rho = 1.34 g/cm3) were compared by Western blot analysis using polyclonal antibodies to HEp-2-grown virus, only traces of protein V (Mr 46,000 Da) could be detected in particles from 293 cells. In contrast, Ad41 particles obtained from HEp-2 cells exhibited a strong band at the position of protein V. Further, if polyclonal antibodies to 293-grown Ad41 were used in the Western blot, no protein V band was detected in HEp-2-grown virus. Finally, we note that new protein bands (Mr 25,000-35,000 Da) could be observed upon Western blot analysis of 293-derived complete and incomplete Ad41 particles. All of these observations taken together suggest that the low infectivity of Ad41 particles, prepared from 293 cells, could be due to a defect in assembly.
Pieniazek
D
D
Department of Microbiology, Immunology, and Parasitology, Louisiana State University Medical Center, New Orleans 70112-1393.
Pieniazek
N J
NJ
Macejak
D
D
Coward
J
J
Rayfield
M
M
Luftig
R B
RB
D000260:Adenoviruses, Human / Q000235:genetics / Q000254:growth & development* / Q000502:physiology; D015153:Blotting, Western; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D003588:Cytopathogenic Effect, Viral; D004279:DNA, Viral / Q000032:analysis / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D015183:Restriction Mapping; D014764:Viral Proteins / Q000032:analysis; D014771:Virion / Q000235:genetics / Q000254:growth & development / Q000502:physiology; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins